PB2171: THE NOVEL CSN5/JAB1 INHIBITOR CSN5I-3 SUPPRESSES CELL GROWTH AND UPREGULATES GENE EXPRESSION OF TYPE-I INTERFERONS (IFN) IN ANAPLASTIC LARGE CELL LYMPHOMA (ALCL)
Authors: I. Xagoraris, G. Kokaraki, K. Stathopoulou, F. X. Claret, E. Drakos, G. Z. Rassidakis
Journal: HemaSphere (2022)

Background: The CSN5/Jab1 (cJun activation domain-binding protein 1), initially discovered as a cJun coactivator, represents the fifth component of an evolutionary highly conserved 8 subunit protein complex, named COP9 signalosome (CSN5). Accumulating evidence suggests that the CSN5/Jab1 gene operates as an oncogene in cancer through multiple mechanisms including cell cycle control via CDK inhibitors. We have previously shown that CSN5/Jab1 protein is overexpressed in T-cell lymphomas with the highest frequency of positive tumors being observed in both ALK+ and ALK- anaplastic large cell lymphomas (ALCL). Targeting CSN5/Jab1 activity in cancer is now possible since a novel inhibitor, namely CSN5i-3 (Novartis), has been developed for clinical use. The potential role of CSN5/Jab1 in modulation of anti-tumor immune responses in cancer, and particularly in ALCL, is unknown to date. Aims: To investigate the effects of the selective CSN5i-3 inhibitor in cell growth and type-I interferon - associated anti-tumor immune responses in ALCL. Methods: The ALCL in vitro system included 5 cell lines (3 ALK+ and 2 ALK- ALCL cell lines). Cell growth and viability were assessed by trypan blue and MTT assays. The effects of the CSN5i-3 on G1-S cell cycle progression were assessed by BrdU incorporation assay and on apoptosis by Annexin V/AAD staining and flow cytometry. Expression of various cell cycle regulating proteins at baseline and experimental conditions were analysed by Westem blot analysis. Gene expression of type I interferons, including interferonbeta (IFN-β), CXCL10 and interferon gamma (IFN-y), as well as, STING and a control gene (GAPDH) at the RNA level, were analysed with qRT-PCR. The ALCL cell lines were treated with the specific CSN5i-3 inhibitor at different concentrations. Silencing of the CSN5/Jab1 gene was performed using transient transfection with specific siRNA construct using the Amaxa Nucleofector System (Lonza). Results: Treatment of ALCL cell lines with increasing concentrations of CSN5i-3 resulted in decreased protein levels of CSN5/Jab1, which was associated with significantly decreased cell growth but slight increase in apoptosis (10-15%). The effects on cell growth linked to downregulation of cell cycle progression were associated with upregulation of the CDK inhibitors p21, p27 and p57. Similarly, CSN5/Jab1 gene silencing resulted in decreased cell growth associated with increased expression of similar cell cycle inhibiting proteins. In addition, inhibition of CSN5/Jab1 activity by CSN5i-3 resulted in increased IFN-β and IFN-y mRNA levels in ALK+ ALCL cells, and CXCL10, IFN-y and STING mRNA levels in ALK- ALCL cells. Similarly, knocking down CSN5/Jab1 gene by transient transfection with siRNA led to upregulation of type-I interferons, particularly IFN-β and CXCL10 in ALCL, suggesting that the effects of CSN5i-3 can be attributed to specific inhibition of CSN5/Jab1 activity. Summary/Conclusion: The novel inhibitor CSN5i-3 sufficiently downregulates expression of CSN5/Jab1 oncogene, inhibits cell cycle progression and contributes to type-I interferon - associated anti-tumor immune response in ALK+ and ALK- ALCL. These preclinical findings suggest a potential role of CSN5i-3 in molecular therapeutics and immunotherapy in T-cell lymphomas.